In this research, the meticulous development and exhaustive analysis of molecularly imprinted polymer nanoparticles (nano-MIPs) are explored, with particular emphasis on their affinity for human serum albumin (HSA), a crucial biomarker for diagnosing type 2 diabetes mellitus. The yield of the synthesized nano-MIPs for HSA was determined by a freeze drying technique to be in the range of 6–15 mg for every 100 g of glass beads. A multifaceted characterization approach, including dynamic light scattering analysis, atomic force microscopy, and Fourier-transform infrared spectroscopy, was employed to rigorously assess the physicochemical properties of the nano-MIPs. The results indicate a relatively uniform size distribution and a well-defined chemical composition. Surface plasmon resonance kinetic analysis further revealed that the nano-MIPs exhibit high affinity and specificity for HSA, with a dissociation constant (Kd) of 2.37 × 10−9 M and an R2 value of 0.97. These findings not only validate the successful synthesis and characterization of nano-MIPs but also underscore their potential use for selective molecular recognition, thereby providing promising avenues for future research and applications in biomedical and environmental fields.